Rat insulin-degrading enzyme: cleavage pattern of the natriuretic

3830 Biochemistry, Vol. 32, No. 14, 1993

Rat Insulin-Degrading Enzyme: Cleavage Pattern of the Natriuretic Peptide Hormones ANP, BNP, and CNP Revealed by HPLC and Mass Spectrometry, by Dieter Muller, Christian Schulze, Hans Baumeister, Friedrich Buck, and Dietmar Richter*, Volume 3 1, Number 45, November 17, 1992, pages 1 1 138-1 1143. Page 1 1 141. In Figure 3, 1251-(Phe-Arg-)should read 1251-(Phe-Arg-Tyr).The correct figure is Leupeptin Phosphoramidon 15 6 0 1 1 5 15 6 0 1 1 5 15 6 0 1 1

H20 1 1

5

PCMB 5 15 601 1

EDTA 5 15 601 m i n i

I__

i

r li

I

-125I-Tyr -125I-(Phe-Arg-Tyr) -1251-(Arg-Tyr) --'251-ANP

FIGURE3: Effect of inhibitors on the degradation of IZ5I-ANPby IDE. Reactions were performed at 25 O C . The enzyme (1.5 ng) was preincubated in 3 pL of 20 mM Hepes, pH 7.5, for 5 min in the presence of either leupeptin (20 pg/mL), phosphoramidon (0.1 mM), p(ch1oromercuri)benzoate (200 pg/mL), or EDTA (10 mM) or in the absence of inhibitors. Subsequently, the substrate (120 pmol of unlabeled ANP and 10 fmol of '2SI-ANP)together with additional inhibitor was added to give a final volume of 12 pL of 20 mM Hepes, pH 7.5, 1 mM DTT, and 5 mM MnCI2. At the times indicated, 2.5 pL was removed, and the reaction was stopped by the addition of 1 pL of a solution containing 25 mM EDTA, 2 mM 1,lO-phenanthroline, and 5 mM N-ethylmaleimide. Samples were analyzed by thin-layer chromatography (Muller et al., 1991). After a run time of 125 min, the sheets were dried and exposed to X-ray films. The positions of '2Wabeled F-R-Y, R-Y, and Y were verified using reference molecules. Chromophore of Sensory Rhodopsin I1 from Hdobacterium halobium, by B. Scharf, B. Hess, and M. Engelhard*, Volume 31, Number 49, December 15, 1992, pages 1248612492. Page 12489. In Table 11, the legend was incompletely reproduced. The table should appear as follows: ~~

Table 11: Purification of Sensory Rhodopsin 11" stage of,purification

protein (mg)

sR-I1 content (nmol)

sR-I1 specific content (nmol/mg of protein)

purification

yield

membrane fraction 2005 72 0.036 0.75 133 solubilized fraction 1830 50 0.027 1 100 butyl-Sepharose fraction 73 38 0.52 19 76 hydroxyapatite fraction 10.5 22 2.1 78 44 gel filtration eluate 3.9 13 3.3 122 26 0 The purification was carried out with material from 20 L of cell culture (strain Dl). Protein content was determined by amino acid analysis after total hydrolyses; sR-I1 content was determined by difference spectroscopy between native and hydroxylamine bleached fraction.